Manipulation of beads in droplets and methods for manipulating droplets

• US 10,139,403 B2
• Filed: 09/15/2016
• Issued: 11/27/2018
• Est. Priority Date: 04/18/2006
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pamula, Vamsee K., Sista, Ramakrishna, Srinivasan, Vijay, Pollack, Michael G., Eckhardt, Allen
• Primary Examiner(s): Sines, Brian J.
• Application Number:
  US15/266,693
• Publication Number:
  US 20170003282A1
• Time in Patent Office: 803 Days
• Field of Search: 422 681, 422502, 422503, 422504, 422 81, 422 82, 436 43, 436174, 436180, 436 63, 436177
• CPC Class Codes: B01F 31/65 the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 33/3031 using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
• CPC Class Codes: B01F 33/451 wherein the mixture is directly exposed to an electromagnetic field without use of a stirrer, e.g. for material comprising ferromagnetic particles or for molten metal
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/06 Auxiliary integrated devices, integrated components
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/043 : magnetic forces
• CPC Class Codes: B01L 2400/0436 : acoustic forces, e.g. surface acoustic waves [SAW]
• CPC Class Codes: B01L 2400/0442 : thermal energy, e.g. vaporisation, bubble jet
• CPC Class Codes: B01L 3/50273 : characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502792 : for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 7/525 : with physical movement of samples between temperature zones
• CPC Class Codes: G01N 1/38 : Diluting, dispersing or mixing samples
• CPC Class Codes: G01N 2001/386 : Other diluting or mixing processes
• CPC Class Codes: G01N 2035/1046 : Levitated, suspended drops
• CPC Class Codes: G01N 33/54326 : Magnetic particles
• CPC Class Codes: Y10T 436/11 : Automated chemical analysis
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/25625 : Dilution
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

The invention provides a method of circulating magnetically responsive beads within a droplet in a droplet actuator. The invention also provides methods for splitting droplets. The invention, in one embodiment, makes use of a droplet actuator with top and bottom substrates, a plurality of magnetic fields respectively present proximate the top and bottom substrates, wherein at least one of the magnet fields is selectively alterable, and a plurality of droplet operations electrodes positioned along at least one of the top and bottom surfaces. A droplet is positioned between the top and bottom surfaces and at least one of the magnetic fields is selectively altered.

Claims

1. A method of manipulating a droplet comprising magnetically responsive beads within a droplet actuator, the method comprising:

(a) providing a droplet actuator, comprising;

(i) a plurality of droplet operations electrodes configured to transport the droplet; and

(ii) a magnetic field present at a portion of the plurality of droplet operations electrodes; and

(b) positioning a magnetic shielding material in the droplet actuator to selectively minimize the magnetic field.

2. A method comprising:

(a) providing a droplet actuator, comprising;

(i) a plurality of reservoir electrodes configured to manipulate a droplet; and

(ii) a plurality of droplet operations electrodes in fluid communication with the plurality of reservoir electrodes; and

(b) operating the plurality of reservoir electrodes to cause beads within a droplet to resuspend within the droplet.

3. The method of claim 2 wherein operating the plurality of reservoir electrodes further comprises randomly activating and deactivating the reservoir electrodes.

4. The method of claim 2 wherein operating the plurality of reservoir electrodes further comprises alternatively and/or concurrently activating and deactivating the reservoir electrodes.

15. The method of claim 2 wherein the beads are magnetically-responsive beads that are aggregated using one or more magnets prior to operating the plurality of reservoir electrodes.

16. The method of claim 15 wherein the one or more magnets comprises a first electromagnet arranged near a top substrate of the droplet actuator and a second electromagnet arranged near a bottom substrate of the electromagnet.

17. The method of claim 2 further comprising the step of apply a high frequency AC voltage to one or more of the reservoir electrodes.

18. The method of claim 17 wherein the high frequency AC voltage is in the range of about 100 volts to about 300 volts with a frequency in the range of about 10 Hz to about 1000 Hz.

19. The method of claim 2 wherein the re-suspending of beads within the droplet occurs in a reservoir of the droplet actuator.

20. The method of claim 2 further comprising dispensing bead-containing droplets when a desired degree of resuspension is achieved based on a target percentage of variation in each droplet.

21. The method of claim 20 wherein the dispensing of bead-containing droplets results in at least 95% consistency in bead count.

5. A method of re-suspending bead within a droplet in a droplet actuator, the method comprising:

(a) providing a droplet actuator, comprising;

(i) a reservoir electrode configured to manipulate a droplet; and

(ii) a plurality of droplet operations electrodes in fluid communication with the reservoir electrode;

(b) separating a slug of the droplet from the droplet on the reservoir electrode; and

(c) recombining the slug with the droplet at the reservoir electrode.

6. The method of claim 5 further comprising transporting the slug along the plurality of droplet operations electrodes.

7. The method of claim 5 further comprising repeating steps (b) and (c).

8. A method of manipulating a droplet comprising magnetically responsive beads within a droplet actuator, the method comprising:

(a) providing a droplet actuator comprising;

(i) a plurality of droplet operations electrodes configured to transport the droplet; and

(ii) a magnetic field present at a portion of the plurality of droplet operations electrodes; and

(b) positioning a plurality of magnets so as to selectively minimize the magnetic field.

9. The method of claim 8 wherein the magnetic field is sufficiently strong to hold the magnetically responsive beads substantially immobile during a droplet operation.

10. The method of claim 8 wherein the magnetic field is sufficiently weak to enable the magnetically responsive bead to be moved away from the magnetic field during a droplet operation.

11. A method of splitting a droplet into two or more sub-droplets, the method comprising:

(a) providing a droplet actuator comprising;

(i) a droplet operations surface comprising droplet operations electrodes, the droplet operations electrodes including one or more intermediate electrodes;

(ii) a top substrate separated from the droplet operations surface to form a gap;

(iii) a physical barrier extending into the gap and constricting the gap in proximity with the one or more intermediate electrodes; and

(iv) a droplet on one or more of the droplet operations electrodes;

(b) activating three or more of the droplet operations including one or more of the intermediate electrodes to elongate the droplet;

(c) deactivating one or more of the intermediate electrodes to cause the droplet to split into two sub-droplets.

12. The method of claim 11 wherein the barrier is mounted on, and extends into the gap from, the top substrate.

13. The method of claim 11 wherein the barrier produces a magnetic field.

14. The method of claim 11 wherein:

(a) the droplet comprises beads or particles;

(b) the barrier restricts passage of the beads or particles; and

(c) the method yields;

(i) a first sub-droplet comprising substantially all of the beads or particles; and

(ii) a second droplet substantially lacking in beads or particles.